Electrostatic interactions impact every aspect of the structure and function of proteins, nucleic acids and membranes. The time-averaged electric fields in macromolecular systems can arise from the organized distribution of charged, polar and polarizable groups, the concentration and mobility of ions in the surrounding solvent, and, in the case of membrane associated processes, transmembrane potentials. The solvation response to charge movement also depends upon this distribution of groups constrained by the structure of the macromolecular assembly. The magnitudes of the electric fields in proteins and the variations in these fields at different sites can be enormous. These variations and their absolute magnitudes are well appreciated by theorists who have developed a large body of analytical, computational, and graphical methods to evaluate electrostatic potentials. However, it has proven to be more difficult to obtain quantitative experimental information on local variations in electric fields in proteins and the time-dependent changes in the electric fields in response to changes in charge. This proposal outlines new types of experiments that probe the time-averaged static field (Aim 1) and solvation response dynamics (Aim 2) in proteins. On-going work in these areas led to important discoveries about the excited-state dynamics in green fluorescent protein (GFP), and extensions of this work form the basis of Aim 3. Aim 1 - Electric fields in proteins. We propose to use vibrational Stark effect spectroscopy to calibrate the sensitivity of specific molecular probe vibrations to electric fields. These oscillators can be displayed on inhibitors, non-natural amino acids or site-specific labels. Once calibrated, measured shifts in vibrational frequencies when the protein is changed or the probe is moved can be used to read out changes in electric fields. We will use human aldose reductase as a model system because of its great importance as a target for the control of diabetes, and because ultrahigh resolution x-ray structures, high-level electrostatics calculations and inhibitors that display useful electric field probes are readily available. Measured electric field changes in response to amino acid perturbations or at different sites will be directly compared with electrostatics calculations to provide a stringent test for these calculations. Aim 2 - Solvation dynamics in proteins. We propose to measure the time-dependent solvation of charge at many sites in a small protein by using novel fluorescent and phosphorescent amino acids and measurements of the dynamic Stokes shift. The time dependence can be directly compared with simulations to dissect the origins of the widely different time scales and differences in the capacity to solvate charge observed at different sites in proteins. Aim 3 - Excited state dynamics in GFP. We propose to expand our understanding of excited-state proton transfer dynamics in GFP mutants, capitalizing on discoveries made during the last grant period. Experiments are proposed on GFPs that emit at multiple wavelengths (dual emission GFPs), on the mechanism of communication between the chromophore and bulk solvent, and on GFP chromophores containing non-natural amino acids. [unreadable] [unreadable]